Modified RNA polymerase sigma factor 70 polypeptide

ABSTRACT

The present invention relates to a novel variant RNA polymerase sigma factor 70 (π70) polypeptide, a polynucleotide encoding the same, a microorganism containing the polypeptide, and a method for producing L-threonine by using the microorganism.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a U.S. national phase application of InternationalPCT Patent Application No. PCT/KR2015/009381, which was filed on Sep. 4,2015, which claims priority to Korean Patent Application Nos.10-2015-0125440, filed Sep. 4, 2015 and 10-2014-0119138, filed Sep. 5,2014 These applications are incorporated herein by reference in theirentireties.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is HANO_047_00US_SeqList_ST25.txt. The text file is166 KB, was created on Aug. 5, 2016, and is being submittedelectronically via EFS-Web.

TECHNICAL FIELD

The present invention relates to a novel variant RNA polymerase sigmafactor 70 (σ⁷⁰) polypeptide, a polynucleotide encoding the same, amicroorganism containing the polypeptide, and a method for producingL-threonine by using the microorganism.

BACKGROUND ART

Generally, useful products such as amino acids can be produced by afermentation method using microorganism strains developed via artificialmutation or genetic recombination. In particular, in developingmicroorganism strains for large-scale production of amino acids, it willbe beneficial to discover genetic factors, which are directly/indirectlyinvolved in a higher cascade step of the production, and appropriatelyutilize them to develop microorganism strains capable of producinghigher yields. A representative technology in this regard may be globaltranscription machinery engineering (gTME), which can regulate theexpression of all intracellular genes by causing random mutations onrecruiting protein of RNA polymerase.

RNA polymerase is a macromolecule comprised of five subunits of 2α, β,β′, and ω, and its holoenzymes are expressed as α₂ββ′ω. Along with theseholoenzymes, sigma (σ) factors, which are transcription initiationfactors present in prokaryotes, can allow specific binding of RNApolymerase to promoters, and can be distinguished by their molecularweight. For example, σ⁷⁰ stands for a sigma factor having a molecularweight of 70 kDa (Gruber™, Gross C A, Annu Rev Microbiol. 57: 441-66,2003).

Escherichia coli is known to possess a housekeeping sigma factor σ⁷⁰(RpoD), a nitrogen-limitation sigma factor σ⁵⁴ (RpoN), astarvation/stationary phase sigma factor σ³⁸ (RpoS), a heat shock sigmafactor σ³² (RpoH), a flagellar sigma factor σ²⁸ (RpoF), anextracytoplasmic/extreme heat stress sigma factor σ²⁴ (RpoE), a ferriccitrate sigma factor σ¹⁹ (FecI), etc. These various sigma factors areknown to be activated under different environmental conditions, andthese characterized sigma factors can bind to the promoters of genestranscribed under specific environmental conditions, and therebyregulate the transcription of the genes. Studies on the increase ofproductivity of target materials by allowing random mutations on sigmafactor 70 have been reported (Metabolic Engineering 9. 2007. 258-267),and there is also a study report on the enhanced tyrosine productionusing gTME technology in E. coli (U.S. Pat. No. 8,735,132).

DISCLOSURE Technical Problem

The present inventors, while endeavoring to develop a microorganismcapable of producing L-threonine at an improved concentration withoutgrowth retardation of a host cell, developed a novel modified sigmafactor 70 polypeptide of RNA polymerase, and also discovered that abacterial strain having an improved L-threonine-producing capability canbe developed by introducing the novel modified sigma factor 70polypeptide of RNA polymerase into Escherichia sp. having anL-threonine-producing capability.

Technical Solution

An object of the present invention is to provide a modified polypeptidehaving an activity of RNA polymerase sigma factor 70 of the amino acidsequence of SEQ ID NO: 8 wherein a part of the amino acid issubstituted.

Another object of the present invention is to provide a polynucleotideencoding the polypeptide.

A further object of the present invention is to provide a transformedmicroorganism which includes the polypeptide.

A still further object of the present invention is to provide a methodof producing L-threonine comprising culturing the microorganism; andrecovering L-threonine from the cultured microorganism or a culturemedium thereof.

Advantageous Effects

The present invention enables confirmation of a novel variant of apolypeptide of an RNA polymerase sigma factor 70 capable of upregulatingthe L-threonine-producing capability. Additionally, a microorganismcapable of expressing the modified polypeptide based on the same has anexcellent yield of L-threonine production, and thus the microorganismcan provide convenience in production, and reduction in production costfrom the industrial point of view.

BEST MODE

In an aspect of the above objects, the present invention provides anovel modified polypeptide having an activity of RNA polymerase sigmafactor 70.

As used herein, the term “RNA polymerase sigma factor 70” refers to aprotein σ⁷⁰, one of sigma factors and is called sigma factor D(RpoD).The protein σ⁷⁰ acts as one of transcription initiation factors alongwith RNA polymerase. Sigma factors are involved in the regulation oftranscription by interacting with upstream DNA (UP element) on upstreamof particular promoters and various transcription factors. Inparticular, sigma factor 70 (σ⁷⁰) is a major regulator among E. colisigma factors, which controls most housekeeping genes and core genes,and is known to predominantly act during the exponential phase of E.coli (Jishage M, et al, J Bacteriol 178(18); 5447-51, 1996). Theinformation on sigma factor 70 protein may be obtained from the knowndatabase such as NCBI GenBank, and, for example, it may be a proteinwith the Accession number NP 417539. Specifically, the σ⁷⁰ protein mayinclude an amino acid sequence of SEQ ID NO: 8, but is not limitedthereto, as long as the protein has the same activity as that of the σ⁷⁰protein of the present invention.

As used herein, the term “modified polypeptide” generally refers to awild-type polypeptide wherein a partial or entire amino acid sequence ofthe polypeptide is substituted. In the present invention, it refers to apolypeptide having the activity of sigma factor 70 (σ⁷⁰) of RNApolymerase with an amino acid sequence partially different from that ofthe wild-type, prepared by substituting part of the amino acid sequenceof the wild-type sigma factor 70 (σ⁷⁰), i.e., a sigma factor 70(σ⁷⁰)-modified polypeptide contributing to the enhancement ofL-threonine-producing capability.

Specifically, the modified polypeptide may be a polypeptide having theactivity of RNA polymerase sigma factor 70 of the amino acid sequence ofSEQ ID NO: 8, wherein at least one amino acid at positions of 440 to450; 459; 466; 470 to 479; 484; 495 to 499; 509; 527; 565 to 570; 575 to580; 599; and 612, from the initial methionine as the first amino acid,is substituted with another amino acid. That is, the modifiedpolypeptide may be a polypeptide wherein an amino acid in at least oneof the 45 positions (positions 440 to 450, 459, 466, 470 to 479, 484,495 to 499, 509, 527, 565 to 570, 575 to 580, 599, and 612) may besubstituted with another amino acid. For example, the number of theposition may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, but may not belimited thereto, as long as it has the activity of RNA polymerase sigmafactor 70.

More specifically, the amino acid at position 440, 446, or 448 amongthose at positions 440 to 450; the amino acid at position 474 or 477among those at positions 470 to 479; the amino acid at position 496 or498 among those at positions 495 to 499; the amino acid at position 567or 569 among those at positions 565 to 570; and the amino acid atposition 576 or 579 among those at positions 575 to 580 may besubstituted with another amino acid, but are not limited thereto.

Further more specifically, the amino acid at position 440 may besubstituted with proline (T440P); the amino acid at position 446 withproline (Q446P); the amino acid at position 448 with serine (R448S); theamino acid at position 459 with asparagine (T459N); the amino acid atposition 466 with serine (I466S); the amino acid at position 474 withvaline (M474V); the amino acid at position 477 with glycine (E477G); theamino acid at position 484 with valine (A484V); the amino acid atposition 496 with asparagine (K496N); the amino acid at position 498with arginine (L498R); the amino acid at position 509 with methionine(T509M); the amino acid at position 527 with proline (T527P); the aminoacid at position 567 with valine (M567V); the amino acid at position 569with proline (T569P); the amino acid at position 576 with glycine(N576G); the amino acid at position 579 with arginine (Q579R), leucine(Q579L), threonine (Q579T), isoleucine (Q579I), glycine (Q579G), alanine(Q579A), proline (Q579P), or serine (Q579S); the amino acid at position599 with cysteine (R599C); or the amino acid at position 612 withglycine (D612G), tyrosine (D612Y), threonine (D612T), asparagine(D612N), phenylalanine (D612F), lysine (D612K), serine (D612S), arginine(D612R), or histidine (D612H), or amino acid deletion with a stop codon(D612*), but may not be limited thereto. When the nucleotide issubstituted with a stop codon there may be no amino acid.

Even more specifically, the modified polypeptide may be a polypeptidehaving an amino acid sequence among the SEQ ID NOS: 9 to 37, but may notbe limited thereto.

The modified polypeptide of the present invention may include not onlythe amino acid sequences of SEQ ID NOS: 9 to 37, but also those having ahomology of at least 70% with these sequences, specifically at least80%, more specifically at least 90%, and even more specifically at least99%, without limitation, as long as the protein can contribute to theenhancement of L-threonine-producing capability, compared to thewild-type sigma factor 70 (σ⁷⁰).

As a sequence having a homology as such, if the amino acid sequence isone which has substantially the same or corresponding biologicalactivity of the modified sigma factor 70 (σ⁷⁰), it is obvious that aminoacid sequences with a deletion, a modification, a substitution, or anaddition in part of the sequences should also be included in the scopeof the present invention.

As used herein, the term “homology” refers to a degree of identity ofnucleotides or amino acid residues between two different amino acidsequences or nucleotide sequences of a gene encoding a protein, asaligning them to be maximally matched in a particular region. When thereis a sufficiently high homology between them, the expression products ofthe corresponding gene may have the same or similar activities. Thehomology between sequences may be determined by a technology known inthe art, for example, known sequence comparison programs including BLAST(NCBI), CLC Main Workbench (CLC bio), MegAlign (DNASTAR Inc), etc.

In another aspect, the present invention provides a polynucleotideencoding the modified polypeptide.

As used herein, the term “polynucleotide” refers to a polymer ofnucleotides, in which nucleotide monomers are connected lengthwise in achain shape by covalent bonds, specifically a DNA or RNA strand. Morespecifically, in the present invention, it may be a polynucleotidefragment encoding the modified polypeptide.

In an exemplary embodiment of the present invention, the gene encodingthe amino acid sequence of RNA polymerase sigma factor 70 is rpoD gene,and may be specifically a gene derived from the genus Escherichia, andmore specifically a gene derived from E. coli. The polynucleotideencoding the wild-type RNA polymerase sigma factor 70 may be representedby SEQ ID NO: 7, but is not limited thereto. Additionally, based on thegenetic code degeneracy, polynucleotide sequences encoding the sameamino acid sequence and variants thereof should also be included in thescope of the present invention.

Additionally, as for the modified polynucleotide of the presentinvention, based on the genetic code degeneracy, polynucleotidesequences encoding the same amino acid sequence and variants thereofshould also be included in the scope of the present invention.Specifically, a nucleotide sequence encoding the polypeptide of theamino acid sequence of SEQ ID NO: 8, wherein at least one amino acid issubstituted with another amino acid described above, or a variantthereof, may be included. In particular, the positions of the abovevariations may be the positions of amino acids at 440 to 450; 459; 466;470 to 479; 484; 495 to 499; 509; 527; 565 to 570; 575 to 580; 599; and612, from the initial methionine as the first amino acid.

More specifically, the positions of the above variations may be asubstitution of the amino acid at position 440 with proline (T440P); asubstitution of the amino acid at position 446 with proline (Q446P); asubstitution of the amino acid at position 448 with serine (R448S); asubstitution of the amino acid at position 459 with asparagine (T459N);a substitution of the amino acid at position 466 with serine (I466S); asubstitution of the amino acid at position 474 with valine (M474V); asubstitution of the amino acid at position 477 with glycine (E477G); asubstitution of the amino acid at position 484 with valine (A484V); asubstitution of the amino acid at position 496 with asparagine (K496N);a substitution of the amino acid at position 498 with arginine (L498R);a substitution of the amino acid at position 509 with methionine(T509M); a substitution of the amino acid at position 527 with proline(T527P); a substitution of the amino acid at position 567 with valine(M567V); a substitution of the amino acid at position 569 with proline(T569P); a substitution of the amino acid at position 576 with glycine(N576G); a substitution of the amino acid at position 579 with arginine(Q579R), leucine (Q579L), threonine (Q579T), isoleucine (Q579I), glycine(Q579G), alanine (Q579A), proline (Q579P), or serine (Q579S); asubstitution of the amino acid at position 599 with cysteine (R599C); ora substitution of the amino acid at position 612 with glycine (D612G),tyrosine (D612Y), threonine (D612T), asparagine (D612N), phenylalanine(D612F), lysine (D612K), serine (D612S), arginine (D612R), or histidine(D612H); or a substitution of nucleotides with a stop codon (D612*), anda nucleotide sequence encoding the amino acid sequence of a modifiedpolypeptide, wherein the amino acid substitution is a combination of atleast one kind among the 34 amino acid substitutions described above, ora variant thereof, may be included.

Even more specifically, a nucleotide sequence encoding any amino acidsequence of the amino acid sequences of SEQ ID NOS: 9 to 37, or avariant thereof, may be included.

In another aspect, the present invention provides a host cell includingthe polynucleotide encoding the modified polypeptide, a microorganismtransformed with a vector including the polynucleotide encoding themodified polypeptide, or a microorganism introduced with the modifiedpolypeptide. Specifically, the introduction may be performed bytransformation, but is not limited thereto.

Specifically, the microorganisms including the sigma factor 70(σ⁷⁰)-modified polypeptide may have enhanced L-threonine-producingcapability without growth inhibition of a host cell, compared to themicroorganism including the wild-type sigma factor 70 (σ⁷⁰) polypeptide,and thus L-threonine can be obtained in high yield from thesemicroorganisms.

As used herein, the term “vector” refers to any mediator for cloningand/or transfer of a nucleotide sequence into a host cell. The vectormay be a replicon to which a different DNA fragment can bind, leading toreplication of a combined fragment. As used herein, the term “replicon”refers to any genetic unit (e.g., plasmids, phages, cosmids,chromosomes, and viruses) which can be replicated by self-regulation.The vector may include viral- or non-viral mediators for in-vivo,ex-vivo, or in-vitro introduction of a nucleotide into a host cell, andmay also include minicircle DNA. For example, the vector may includeplasmids which do not have any bacterial DNA sequence (Ehrhardt, A. etal. (2003) HumGene Ther 10: 215-25; Yet, N. S. (2002) MoI Ther 5:731-38; Chen, Z. Y. et al. (2004) Gene Ther 11: 856-64). Additionally,the vector may include transposons (Annu Rev Genet. 2003; 37: 3-29.), orartificial chromosomes. Specifically, pACYC177, pACYC184, pCL1920,pECCG117, pUC19, pBR322, pDZ, pCC1BAC, and pMW118 vectors may be used,but they are not limited thereto.

As used herein, the term “transformation” refers to introducing a geneinto a host cell to be expressed in the host cell, and the transformedgene may not be particularly limited as long as it can be expressed inthe host cell, regardless of whether the transformed gene is insertedinto the chromosome of the host cell or positioned outside of thechromosome.

The gene may be introduced into a host cell in the form of an expressioncassette, which is a polynucleotide construct including all essentialelements for self-expression. The expression cassette may include apromoter, which is conventionally operably connected to the gene, atranscription termination signal, a ribosome-binding domain, and atranslation termination signal. The expression cassette may be aself-replicable expression vector. Additionally, the gene may be onewhich is introduced into a host cell as a gene itself or in the form ofa polynucleotide construct to be connected to a sequence necessary to beexpressed in a host cell, but is not limited thereto.

As used herein, the term “host cell” or “microorganism” may refer to anycell or microorganism which includes a polynucleotide encoding amodified polypeptide, or which is transformed by a vector including thepolynucleotide encoding a modified polypeptide and thus can express themodified polypeptide.

In the present invention, the host cell or microorganism may be any cellor microorganism capable of producing L-threonine and including themodified sigma factor 70 (σ⁷⁰). Examples of the microorganism mayinclude Escherichia sp., Serratia sp., Erwinia sp., Enterobacteria sp.,Salmonella sp., Streptomyces sp., Pseudomona sp., Brevibacterium sp.,Corynebacteria sp., etc.; and specifically, a microorganism belonging toEscherichia sp., and more specifically, Escherichia coli, but it is notlimited thereto.

In another aspect, the present invention provides a method of producingL-threonine including culturing the described microorganism in a medium,and recovering L-threonine from the cultured microorganism or theculture medium thereof.

As used herein, the term “culturing” refers to growing the microorganismin an appropriately and artificially adjusted environment. The cultureprocess may be performed according to the appropriate medium andconditions for culture known in the art. The specific culturing processmay be performed according to the general knowledge of one of ordinaryskill in the art or the conventional method known in the art, and may beappropriately adjusted accordingly. Specifically, the culturing methodsare described in detail in [Chmiel; Bioprozesstechnik 1. Einfuhrungindie Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991), andStorhas; Bioreaktoren and periphere Einrichtungen (Vieweg Verlag,Braunschweig/Wiesbaden, 1994)]. Additionally, the culturing methods mayinclude a batch culture, a continuous culture, and a fed-batch culture,and specifically, may be cultured continuously in a fed batch orrepeated fed batch process, but are not limited thereto.

The culture medium used for cultivation should meet the requirements foreach specific strain. Examples of the carbon source to be contained inthe medium may include saccharides and carbohydrates such as glucose,sucrose, lactose, fructose, maltose, starch, and cellulose; oils andfats such as soybean oil, sunflower oil, castor oil, and coconut oil;fatty acids such as palmitic acid, stearic acid, and linoleic acid;alcohols such as glycerol and ethanol; and organic acids such as aceticacid. These carbon sources may be used alone or in combination, but arenot limited thereto. Examples of the nitrogen source to be contained inthe medium may include peptone, yeast extract, gravy, malt extract, cornsteep liquor, and bean flour, urea or inorganic nitrogen sources such asammonium sulfate, ammonium chloride, ammonium phosphate, ammoniumcarbonate, and ammonium nitrate. These nitrogen sources may be usedalone or in combination, but are not limited thereto. Examples of thephosphorous source to be contained in the medium may include potassiumdihydrogen phosphate, dipotassium hydrogen phosphate, and correspondingsodium-containing salts, but are not limited thereto. The culture mediamay include metals such as magnesium sulfate and iron sulfate.Additionally, materials essential for growth such as amino acids andvitamins may also be included. Additionally, precursors suitable for themedium may also be used. These materials may be added to the culture inthe form of a batch culture or continuous culture, but are not limitedthereto.

Additionally, the pH of the culture may be adjusted by adding a compoundsuch as ammonium hydroxide, potassium hydroxide, ammonia, phosphoricacid, and sulfuric acid during cultivation in an appropriate manner.Additionally, bubble formation may be prevented during the cultivationusing an antifoaming agent such as fatty acid polyglycol ester.Additionally, oxygen gas or an oxygen-containing gas may be added to aculture in order to maintain aerobic conditions in a culture liquid; noair may be added to maintain anaerobic conditions or microaerobicconditions; or nitrogen gas, hydrogen gas, or carbon dioxide may beinjected. The cultivation may be performed at 27° C. to 37° C., andspecifically at 30° C. to 35° C. The cultivation may be continued untilthe desired amount of production of a useful material can be obtained,and specifically for 10 hours to 100 hours. L-Threonine may be exportedinto a culture medium or may remain contained in the microorganism.

The method of recovering L-threonine from the microorganism or a culturethereof is widely known in the art. For example, methods such asfiltration, anion exchange chromatography, crystallization, HPLC, etc.may be used, but are not limited thereto.

MODE FOR INVENTION

Hereinafter, the present invention will be described in more detail withreference to the following Examples. However, these Examples are forillustrative purposes only, and the invention is not intended to belimited by these Examples.

Example 1 Construction of a Recombinant Vector pCC1BAC-rpoD

In order to obtain a DNA fragment with a size of about 2.0 kb includingthe rpoD gene (NCBI Gene ID: 947567, SEQ ID NO: 7), the chromosomal DNA(gDNA) of Escherichia coli wild-type strain W3110 was extracted usingGenomic-tip System (Qiagen), and a polymerase chain reaction (“PCR”,hereinafter) was performed using the gDNA as a template with a PCR HLpremix kit (BIONEER, Korea; the same product was used hereinafter).

A PCR reaction to amplify the rpoD gene was performed using primers SEQID NO: 1 and SEQ ID NO: 2 by denaturing at 95° C. for 30 seconds,annealing at 56° C. for 30 seconds, and elongation at 72° C. for 2minutes for 27 cycles. The PCR products were digested with HindIII andEcoRI, electrophoresed on a 0.8% agarose gel, and a 2.0 kb DNA fragment(“rpoD fragment”, hereinafter) was obtained therefrom by elution.

TABLE 1 Primer No. Nucleotide Sequence SEQ ID NO 1 5′-TACTCAAGCTTCGGCT 1TAAGTGCCGAAGAGC-3′ 2 5′-AGGGCGAATTCCTGAT 2 CCGGCCTACCGATTA-3′

Subsequently, the Copycontrol pCC1BAC vector (EPICENTRE, USA) wasdigested with HindIII and EcoRI, electrophoresed on a 0.8% agarose gel,and obtained therefrom by elution. The resultant was ligated to the rpoDfragment to construct the pCC1BAC-rpoD plasmid.

Example 2 Construction of a Recombinant Vector pCC1BAC-Partial rpoD

In order to obtain a DNA fragment with a size of about 1.5 kb includingthe region from the promoter to the BamHI restriction site within therpoD gene of E. coli W3110, PCR was performed using the gDNA prepared inExample 1 as a template.

The PCR reaction was performed using primers SEQ ID NO: 1 and SEQ ID NO:3 by denaturing at 95° C. for 30 seconds, annealing at 56° C. for 30seconds, and elongation at 72° C. for 1 minute and 30 seconds for 27cycles as in Example 1. The PCR products were digested with BamHI andHindIII, electrophoresed on a 0.8% agarose gel, and a 1.5 kb DNAfragment was obtained therefrom by elution.

TABLE 2 Primer No. Nucleotide Sequence SEQ ID NO 1 5′-TACTCAAGCTTCGGCT 1TAAGTGCCGAAGAGC-3′ 3 5′-GACGGATCCACCAGGT 3 TGCGTA-3′

Subsequently, the Copycontrol pCC1BAC vector was digested with BamHI andHindIII, electrophoresed on a 0.8% agarose gel, and obtained by elution.The resultant was ligated to the partial rpoD fragment to construct thepCC1BAC-patial rpoD plasmid.

Example 3 Generation of rpoD^(m) Fragment Via Error-Prone PCR

In order to introduce a random modification in the conserved regions2.4, 3, and 4 of the rpoD gene of W3110, the inventors intended toobtain a DNA pool of rpoD fragments, in which random modifications wereintroduced from the BamHI restriction site within the gene to theterminus encoding the gene.

To this end, a PCR reaction was performed using the gDNA obtained inExample 1 with a Diversify PCR Random Mutagenesis kit (catalog #:630703; Clonetech), according to the conditions for mutagenesisreactions 4 in Table III described in the User Manual thereof.Specifically, the PCR was performed using the primers of SEQ ID NO: 2and SEQ ID NO: 4, by denaturing at 94° C. for 30 seconds, annealing at56° C. for 30 seconds, and elongation at 68° C. for 30 seconds for 25cycles.

TABLE 3 Primer No. Nucleotide Sequence SEQ ID NO 2 5′-AGGGCGAATTCCTGAT 2CCGGCCTACCGATTA-3′ 4 5′-AACCTGGTGGATCCGT 4 CAGGCGATC-3′

Subsequently, the mutated art rpoD DNA pool, in which random nucleotidesubstitutions were introduced, was obtained as a PCR product, and thePCR product was digested with BamHI and EcoRI, electrophoresed on a 0.8%agarose gel, and a 0.5 kb DNA fragment (“art rpoD fragment”,hereinafter) was obtained therefrom by elution.

Example 4 Construction of a Recombinant Vector pCC1BAC-rpoD MutantLibrary Including Modified rpoD

The pCC1BAC-partial rpoD vector constructed in Example 2 was treatedwith BamHI and EcoRI, and then treated with alkaline phosphatase (NEB).

Then, the art rpoD fragments obtained in Example 3 were treated withBamHI and EcoRI, respectively, and ligated to the pCC1BAC-partial rpoDvector, which was already treated with the restriction enzymes,transformed into TransforMax EPI300 Electrocompetent E. coli(EPICENTRE), cultured in an LB plate containing 15 μg/mL ofchloramphenicol, and colonies were selected therefrom. The selectedcolonies were collected and subjected to a plasmid prep to construct apCC1BAC-rpoD mutant library.

Example 5 Introduction of a pCC1BAC-rpoD Mutant Library into aThreonine-Producing Strain

The pCC1BAC-rpoD mutant library constructed in Example 4 was introducedinto an electrocompetent cell of KCCM10541, which is athreonine-producing strain, by transformation.

In particular, the KCCM10541 (Korean Patent No. 10-0576342), the E. colistrain used in this Example, is an E. coli strain derived from theKFCC10718 (Korean Patent No. 10-0058286), in which galR gene isinactivated.

Example 6 Comparison of L-Threonine Producing Capabilities BetweenRecombinant Microorganisms and Confirmation of Nucleotide Sequences

The recombinant microorganism library constructed in Example 5 wascultured in titer medium shown in Table 4 below, and the improvement inL-threonine production was examined.

TABLE 4 Composition Conc. (per 1 L) Glucose 70 g KH₂PO₄ 2 g (NH₄)₂SO₄ 25g MgSO₄•7H₂O 1 g FeSO₄•7H₂O 5 mg MnSO₄•4H₂O 5 mg DL-methionine 0.15 gYeast extract 2 g Calcium carbonate 30 g pH 6.8

Specifically, E. coli KCCM10541/pCC1BAC-rpoD and E. coliKCCM10541/pCC1BAC-rpoD mutant library, which were cultured overnight ina solid LB medium in a 33° C. incubator, were inoculated into 25 mL oftiter medium by a platinum loop, respectively, and cultured in a 33° C.incubator while shaking at 200 rpm for 48 hours. The whole procedure wasrepeated to evaluate the rpoD mutant library, and those clones withimproved yield were selected.

TABLE 5 Increase rate of L-Threonine L-threonine Position of Strain(g/L) Conc. (%) modification SEQ ID NO KCCM 10541 (parent strain) 30.4 —KCCM 30.4 — 8 10541/pCC1BAC-rpoD KCCM 32.8 7.9 579, 910541/pCC1BAC-rpoD^(m1) 612 KCCM 33.0 8.6 579, 1010541/pCC1BAC-rpoD^(m2) 612 KCCM 33.6 10.5 579, 1110541/pCC1BAC-rpoD^(m3) 612 KCCM 34.0 11.8 579, 1210541/pCC1BAC-rpoD^(m4) 612 KCCM 33.4 9.9 579, 1310541/pCC1BAC-rpoD^(m5) 612 KCCM 34.0 11.8 579, 1410541/pCC1BAC-rpoD^(m6) 612 KCCM 33.5 10.2 579, 1510541/pCC1BAC-rpoD^(m7) 612 KCCM 32.5 6.9 579, 1610541/pCC1BAC-rpoD^(m8) 612 KCCM 32.0 5.3 579, 1710541/pCC1BAC-rpoD^(m9) 612 KCCM 32.0 5.3 579, 1810541/pCC1BAC-rpoD^(m10) 612 KCCM 32.1 5.6 579, 1910541/pCC1BAC-rpoD^(m11) 612 KCCM 32.0 5.3 579, 2010541/pCC1BAC-rpoD^(m12) 612 KCCM 34.0 11.8 579, 2110541/pCC1BAC-rpoD^(m13) 612 KCCM 34.2 12.6 440 2210541/pCC1BAC-rpoD^(m14) KCCM 34.0 11.8 440, 23 10541/pCC1BAC-rpoD^(m15)496 KCCM 32.4 6.6 446, 24 10541/pCC1BAC-rpoD^(m16) 448, 466, 527, 567KCCM 32.5 7.1 440, 25 10541/pCC1BAC-rpoD^(m17) 477, 498 KCCM 31.9 4.8440, 26 10541/pCC1BAC-rpoD^(m18) 599 KCCM 33.8 11.3 440, 2710541/pCC1BAC-rpoD^(m19) 484 KCCM 34.0 11.9 459, 2810541/pCC1BAC-rpoD^(m20) 474, 509 KCCM 31.9 4.8 440, 2910541/pCC1BAC-rpoD^(m21) 576 KCCM 33.9 11.6 440, 3010541/pCC1BAC-rpoD^(m22) 569

The result revealed, as shown in Table 5 above, that the parent strainKCCM 10541 and the control strain KCCM 10541/pCC1BAC-rpoD produced about30.4 g/L of L-threonine when cultured for 48 hours.

In contrast, the recombinant E. coli introduced with the pCC1BAC-rpoDmutant library produced L-threonine ranging from 31.9 g/L to 34.2 g/L,thus showing an improved L-threonine-producing capability, compared toits parent strain, i.e., an improvement of 4.8% to 12.6% inL-threonine-producing capability compared to its parent strain.

Additionally, the position of modification and the substituted aminoacid in each modification of modified rpoD gene of E. coli with improvedL-threonine-producing capability were examined by sequencing, and theresults are shown in Table 5.

Meanwhile, the recombinant E. coli with the most improvement inL-threonine-producing capability among the transformed E. coli,designated as “KCCM10541/pCC1BAC-rpoD^(m19)”, was deposited on Aug. 6,2014, at the Korean Culture Center of Microorganisms (Accession No:KCCM11560P).

Example 7 Construction of a Wild-Type Strain Introduced with SelectedrpoD Variants and a Wild-Type Strain with Enhanced Biosynthesis Pathwayfor Threonine Production Thereto

A few variations among the rpoD variants, which were confirmed withtheir improved threonine-producing capabilities in Example 6, weresubjected to reconfirm their effects based on wild-type strains. Thewild-type strain W3110 was transformed with the rpoD variationsconfirmed in Example 6 in the same manner as in Example 5, and wasassigned as W3110/pCC1BAC-rpoD^(m). The strain introduced with the rpoDvariation was introduced with pACYC184-thrABC vector to provide thestrain with a threonine-producing capability. The pACYC184-thrABC wasconstructed as described below.

PCR was performed using the genomic DNA of an L-threonine-producing E.coli strain KCCM 10541 (Korean Patent No. 10-0576342; Chinese Patent No.100379851C) derived from E. coli strain KCCM 10718 (Korean Patent No.10-0058286) as a template along with primers of SEQ ID NOS: 5 and 6(Table 6). The DNA fragments obtained therefrom were separated/purified,prepared by treating with HindIII followed by purification, and therebythrABC DNA fragments were prepared. The pACYC184 vector was prepared bytreating with HindIII followed by purification, and ligated to therebyconstruct a pACYC184-thrABC vector. The thus-prepared vector wasintroduced into the W3110/pCC1BAC-rpoD^(m) strain to construct aW3110/pCC1BAC-rpoD^(m), pACYC184-thrABC strain.

TABLE 6 SEQ ID NO Primer Sequence 5 5′-CGAGAAGCTTAGCTTTTCATTCTGACTGCA-3″6 5′-CGAGAAGCTTATTGAGATAATGAATAGATT-3′

Example 8 Comparison of L-Threonine-Producing Capabilities Between aWild-Type Strain, a Wild-Type Strain-Based Recombinant Microorganismwith rpoD Variations, and the Strain with Enhanced Biosynthesis Pathwayfor Threonine Production Thereto

The recombinant microorganisms prepared in Example 7 were cultured in anErlenmeyer flask using a threonine titer medium, and its improvedL-threonine productivity was thereby confirmed.

TABLE 7 Composition Conc. (per 1 L) Glucose 70 g KH₂PO₄ 2 g (NH₄)₂SO₄ 25g MgSO₄•7H₂O 1 g FeSO₄•7H₂O 5 mg MnSO₄•4H₂O 5 mg Yeast extract 2 gCalcium carbonate 30 g pH 6.8

A platinum loop of each of the W3110/pCC1BAC-rpoD^(m),W3110/pACYC184-thrABC, pCC1BAC, and W3110/pACYC184-thrABC,pCC1BAC-rpoD^(m) strains cultured overnight in a solid LB medium in a33° C. incubator was inoculated a titer medium (25 mL) shown in Table 7,and cultured in a 33° C. incubator at the rate of 200 rpm for 48 hours.The results are shown in Table 8 below.

TABLE 8 Glucose Consumption L-Threonine Yield Strain OD (g/L) (g/L) (%)W3110/pCC1BAC 15.4 52.2 0 0 W3110/pCC1BAC-rpoD 15.4 52.2 0 0W3110/pCC1BAC-rpoD^(m2) 15.0 50.6 0 0 W3110/pCC1BAC-rpoD^(m19) 15.5 52.00 0 W3110/pACYC184-thrABC, 13.4 50.1 1.42 2.8 pCC1BACW3110/pACYC184-thrABC, 13.3 50.2 1.43 2.8 pCC1BAC-rpoDW3110/pACYC184-thrABC, 12.5 51.2 1.52 3.0 pCC1BAC-rpoD^(m2)W3110/pACYC184-thrABC, 11.2 51.0 1.56 3.1 pCC1BAC-rpoD^(m19)

As shown in Table 8, the wild-type strain W3110/pCC1BAC and otherstrains of W3110/pCC1BAC-rpoD, W3110/pCC1BAC-rpoD^(m2), andW3110/pCC1BAC-rpoD^(m19) did not produce L-threonine at all when theywere cultured for 48 hours, whereas the strains introduced with variantsshowed a decrease in glucose consumption. The W3110/pACYC184-thrABC,pCC1BAC strain, which is a recombinant strain constructed for producingL-threonine in a wild-type base, produced 1.42 g/L of L-threonine, andthe W3110/pACYC184-thrABC, pCC1BAC-rpoD strain produced 1.43 g/L ofL-threonine, thus showing a 2.8% yield.

In contrast, the W3110/pACYC184-thrABC, pCC1BAC-rpoD^(m2) strain and theW3110/pACYC184-thrABC, pCC1BAC-rpoD^(m19) strain, which arewild-type-based recombinant strains introduced with the rpoD variations,respectively showed glucose consumption for 48 hours in the amount of51.2 g/L and 51.0 g/L, and respectively produced threonine in the amountof 1.50 g/L and 1.53 g/L, thus showing 3.0% and 3.1% yields ofthreonine. That is, it was confirmed that the introduction of the rpoDvariation improved the threonine yield by about 7% to 10%, therebyreconfirming that the rpoD variations selected in the present inventionwere valid variants.

Example 9 Examination of L-Threonine-Producing Capability by theCombination of Selected Recombinant rpoD Variations

In order to examine the changes in threonine-producing capabilities bythe combination of the variations included in each different subjectamong the selected variations, vectors with combined variations wereconstructed for several of the most frequently selected variations. AnrpoD^(m23) (SEQ ID NO: 31) variation, where the variations in amino acidsequences at positions of 440, 579, and 612 were combined, wasconstructed by combining the rpoD variation and the rpoD^(m14) variationevaluated above. Further, an rpoD^(m24) (SEQ ID NO: 32) variation, whichwas introduced with the most variations, was constructed by combiningthe rpoD^(m16) variation and the rpoD^(m3) variation. The rpoD^(m24)variation was introduced with both the rpoD^(m16) variation, which arevariations in amino acid sequences at positions of 446, 448, 466, 527,and 567, and the rpoD^(m3) variation in amino acid sequences atpositions of 579 and 612. Additionally, among the 3 region variations,an rpoD^(m25) (SEQ ID NO: 33) variation was constructed by combining thevariation in the amino acid sequence at position 496 of the rpoD^(m15)and the variations in the amino acid sequence at positions 579 and 612of rpoD^(m1).

Additionally, combinations of amino acid variations present in mutuallydifferent variations were constructed to confirm their effects. Forexample, the amino acid variations at the most frequently selectedpositions of 440, 579, and/or 612 were combined to construct therpoD^(m26) (SEQ ID NO: 34), where the variations at positions 440 and579 were combined; and the rpoD^(m27) (SEQ ID NO: 35), where thevariations at positions 440 and 612 were combined.

Additionally, combinations of low-frequency variations among theselected variations were constructed to confirm their effects. Forexample, to construct the rpoD^(m28) (SEQ ID NO: 36), the variation atposition 477 of the rpoD^(m17), the variation at position 484 of therpoD^(m19), and the variation at position 509 of the rpoD^(m20) werecombined; and to construct the rpoD^(m29) (SEQ ID NO: 37), the variationat position 599 of the rpoD^(m18), the variation at position 459 of therpoD^(m20), and the variation at position 576 of rpoD^(m21) werecombined.

The thus-prepared vectors introduced with rpoD^(m23), rpoD^(m24),rpoD^(m25), rpoD^(m26), rpoD^(m27), rpoD^(m28), and rpoD^(m29)variations were introduced into W3110 along with the pACYC184-thrABCvector prepared in Example 7, and titer evaluation was performed usingthe medium shown in Table 7. The results are shown in Table 9 below.

TABLE 9 Glucose Position SEQ Consumption L-Threonine Yield of ID StrainOD (g/L) (g/L) (%) Variation NO W3110/pACYC184-thrABC, 13.2 50.5 1.402.8 pCC1BAC W3110/pACYC184-thrABC, 13.1 50.8 1.44 2.8 pCC1BAC-rpoDW3110/pACYC184-thrABC, 13.6 52.5 1.61 3.1 440, 579, 31pCC1BAC-rpoD^(m23) 612 W3110/pACYC184-thrABC, 12.0 49.5 1.50 3.0 446,448, 32 pCC1BAC-rpoD^(m24) 466, 527, 567, 579, 612W3110/pACYC184-thrABC, 12.9 52.5 1.52 2.9 496, 579, 33pCC1BAC-rpoD^(m25) 612 W3110/pACYC184-thrABC, 13.3 51.4 1.52 3.0 440,579 34 pCC1BAC-rpoD^(m26) W3110/pACYC184-thrABC, 13.9 50.5 1.54 3.0 440,612 35 pCC1BAC-rpoD^(m27) W3110/pACYC184-thrABC, 12.8 48.5 1.39 2.9 477,484, 36 pCC1BAC-rpoD^(m28) 509 W3110/pACYC184-thrABC, 12.6 50.3 1.49 3.0459, 576, 37 pCC1BAC-rpoD^(m29) 599

From the foregoing, a skilled person in the art to which the presentinvention pertains will be able to understand that the present inventionmay be embodied in other specific forms without modifying the technicalconcepts or essential characteristics of the present invention. In thisregard, the exemplary embodiments disclosed herein are only forillustrative purposes and should not be construed as limiting the scopeof the present invention. On the contrary, the present invention isintended to cover not only the exemplary embodiments but also variousalternatives, modifications, equivalents and other embodiments that maybe included within the spirit and scope of the present invention asdefined by the appended claims.

The invention claimed is:
 1. A modified RNA polymerase sigma factor 70polypeptide, wherein the modified polypeptide has RNA polymerase sigmafactor 70 activity, wherein the modified polypeptide comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence of SEQ ID NO: 8, wherein an amino acid corresponding toposition 612 of the amino acid sequence of SEQ ID NO: 8 is substitutedwith tyrosine, threonine, asparagine, lysine, serine, arginine, orhistidine in the amino acid sequence of the modified polypeptide;wherein the modified polypeptide is able to increase L-threonineproduction in a microorganism comprising the modified polypeptide; andwherein the modified polypeptide further comprises one to sixmutation(s) of an amino acid corresponding to positions selected from440, 446, 448, 459, 466, 474, 477, 484, 496, 498, 509, 527, 567, 569,576, 579, and 599 of the amino acid sequence of SEQ ID NO:
 8. 2. Themodified polypeptide of claim 1, wherein the amino acid corresponding toposition 440 of SEQ ID NO: 8 is substituted with proline in the aminoacid sequence of the modified polypeptide; wherein the amino acidcorresponding to position 446 of SEQ ID NO: 8 is substituted withproline in the amino acid sequence of the modified polypeptide; theamino acid corresponding to position 448 of SEQ ID NO: 8 is substitutedwith serine in the amino acid sequence of the modified polypeptide; theamino acid corresponding to position 459 of SEQ ID NO: 8 is substitutedwith asparagine in the amino acid sequence of the modified polypeptide;the amino acid corresponding to position 466 of SEQ ID NO: 8 issubstituted with serine in the amino acid sequence of the modifiedpolypeptide; the amino acid corresponding to position 474 of SEQ ID NO:8 is substituted with valine in the amino acid sequence of the modifiedpolypeptide; the amino acid corresponding to position 477 of SEQ ID NO:8 is substituted with glycine in the amino acid sequence of the modifiedpolypeptide; the amino acid corresponding to position 484 of SEQ ID NO:8 is substituted with valine in the amino acid sequence of the modifiedpolypeptide; the amino acid corresponding to position 496 of SEQ ID NO:8 is substituted with asparagine in the amino acid sequence of themodified polypeptide; the amino acid corresponding to position 498 ofSEQ ID NO: 8 is substituted with arginine in the amino acid sequence ofthe modified polypeptide; the amino acid corresponding to position 509of SEQ ID NO: 8 is substituted with methionine in the amino acidsequence of the modified polypeptide; the amino acid corresponding toposition 527 of SEQ ID NO: 8 is substituted with proline in the aminoacid sequence of the modified polypeptide; the amino acid correspondingto position 567 of SEQ ID NO: 8 is substituted with valine in the aminoacid sequence of the modified polypeptide; the amino acid correspondingto position 569 of SEQ ID NO: 8 is substituted with proline in the aminoacid sequence of the modified polypeptide; the amino acid correspondingto position 576 of SEQ ID NO: 8 is substituted with glycine in the aminoacid sequence of the modified polypeptide; the amino acid correspondingto position 579 of SEQ ID NO: 8 is substituted with alanine, arginine,leucine, threonine, isoleucine, glycine, proline, or serine in the aminoacid sequence of the modified polypeptide; or the amino acidcorresponding to position 599 of SEQ ID NO: 8 is substituted withcysteine in the amino acid sequence of the modified polypeptide.
 3. Amethod for producing L-threonine comprising culturing a microorganismcomprising the modified polypeptide of claim 1 in a medium to produceL-threonine; and recovering the L-threonine from the culturedmicroorganism or the culture medium.
 4. A method for producingL-threonine comprising culturing a microorganism comprising the modifiedpolypeptide of claim 2 in a medium to produce L-threonine; andrecovering the L-threonine from the cultured microorganism or theculture medium.
 5. The method for producing L-threonine of claim 3,wherein the microorganism is Escherichia coli.
 6. A modified RNApolymerase sigma factor 70 polypeptide wherein the modified polypeptidecomprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 9 to 21, 32, and
 33. 7. A polynucleotide encoding themodified polypeptide of claim
 1. 8. A microorganism comprising themodified polypeptide of claim
 1. 9. The microorganism of claim 8,wherein the microorganism is Escherichia coli.